Regulation of the murine αB-crystallin gene in lens

R. Gopal-Srivastava, Ales Cvekl, J. Piatigorsky

Research output: Contribution to journalArticle

Abstract

Purpose. Previous experiments identified an upstream muscle-preferred enhancer (-426/-259) and a lens-specific regulatory element (LSR, -147/-118) within the mouse αB-crystallin promoter. The purpose of this study was to identify additional cis-control elements and transcription factors responsible for αB-crystallin gene expression in lens. Methods. The activity of αB-crystallin promoter sequences -115/+44, -68/+44 and -164/+44 (with LSR mutation) fused to the bacterial CAT gene were tested by transgenic mouse experiments and by transient transfections (with a cotransfected Pax-6 expression vector). DNase I footprinting and gel shifts were performed using lens nuclear extract, purified Pax-6 and anti-Pax-6 antibody. Results. Transgenic experiments demonstrated that the αB -115/+44 construct (containing a highly conserved region, called here LSE, -78/-46) expresses CAT exclusively in lens, although to a 30-fold lower level than the αB -164/+44-CAT construct. Further deletion to-68 leads to no expression of CAT in the lens. Footprinting indicated that the LSR and LSE control elements are occupied by proteins present in lens nuclear extract and by purified Pax-6. Antibody-gel shift assays suggested that the lens protein which binds to LSR is antigenically related to Pax-6. Cotransfection experiments demonstrated that Pax-6 stimulates the activity of the -164/+44 and -115/+44/αB promoter-CAT gene in fibroblasts. Conclusions. LSE is sufficient for the lens-specific expression of the αB-crystallin gene. Pax-6 is an important transcription factor involved in regulation of the αB-crystallin gene via LSR and LSE in the lens.

Original languageEnglish (US)
JournalInvestigative Ophthalmology and Visual Science
Volume37
Issue number3
StatePublished - Feb 15 1996
Externally publishedYes

Fingerprint

Crystallins
Lenses
Genes
Transcription Factors
Gels
Bacterial Genes
Antibodies
Deoxyribonuclease I
Transgenic Mice
Transfection
Fibroblasts
Gene Expression
Muscles
Mutation

ASJC Scopus subject areas

  • Ophthalmology

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Regulation of the murine αB-crystallin gene in lens. / Gopal-Srivastava, R.; Cvekl, Ales; Piatigorsky, J.

In: Investigative Ophthalmology and Visual Science, Vol. 37, No. 3, 15.02.1996.

Research output: Contribution to journalArticle

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abstract = "Purpose. Previous experiments identified an upstream muscle-preferred enhancer (-426/-259) and a lens-specific regulatory element (LSR, -147/-118) within the mouse αB-crystallin promoter. The purpose of this study was to identify additional cis-control elements and transcription factors responsible for αB-crystallin gene expression in lens. Methods. The activity of αB-crystallin promoter sequences -115/+44, -68/+44 and -164/+44 (with LSR mutation) fused to the bacterial CAT gene were tested by transgenic mouse experiments and by transient transfections (with a cotransfected Pax-6 expression vector). DNase I footprinting and gel shifts were performed using lens nuclear extract, purified Pax-6 and anti-Pax-6 antibody. Results. Transgenic experiments demonstrated that the αB -115/+44 construct (containing a highly conserved region, called here LSE, -78/-46) expresses CAT exclusively in lens, although to a 30-fold lower level than the αB -164/+44-CAT construct. Further deletion to-68 leads to no expression of CAT in the lens. Footprinting indicated that the LSR and LSE control elements are occupied by proteins present in lens nuclear extract and by purified Pax-6. Antibody-gel shift assays suggested that the lens protein which binds to LSR is antigenically related to Pax-6. Cotransfection experiments demonstrated that Pax-6 stimulates the activity of the -164/+44 and -115/+44/αB promoter-CAT gene in fibroblasts. Conclusions. LSE is sufficient for the lens-specific expression of the αB-crystallin gene. Pax-6 is an important transcription factor involved in regulation of the αB-crystallin gene via LSR and LSE in the lens.",
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